Aminoacyl-tRNA synthetases are enzymes found in all bacteria, plants and animals, and are required for cells to make protein. Bacterial aminoacyl-tRNA synthetases are usually present as a single species for each amino acid; approximately 20 different synthetases are required for aminoacylation. Lacking redundancy in the aminoacylation step of protein synthesis, bacteria are critically dependent on the transcription and translation of each of these enzymes. Inhibitors that block the production or function of one or more bacterial synthetases are therefore potentially useful as antimicrobial agents for prevention and treatment of disease in humans and other organisms.
A potential target for shutting-down expression of bacterial aminoacyl synthetase genes is the T-box termination/antitermination transcriptional control complex. Many Gram positive, and certain Gram negative bacteria have genes that are regulated by a transcriptional control element known as the T-box termination/antitermination complex. This complex comprises a set of specific control elements that are found within the leader sequences located upstream of certain bacterial genes. The structural arrangement of these leaders is conserved across a broad spectrum of bacterial strains, and the control elements that are specific to the T-box termination/antitermination complex are highly conserved. The T-box termination/antitermination complex regulates expression of aminoacyl-tRNA synthetase genes and other amino acid-related genes in response to the level of charging of cognate tRNAs. Uncharged tRNAs permit read-through of the template by favoring the antiterminator configuration, thus resulting in expression of the gene located downstream of the leader region. Charged tRNAs block read-through of the template by favoring the terminator configuration, thus resulting in termination of expression. Since expression of these T-box regulated genes is required for survival of the bacterial cell, and this system is found in many pathogenic organisms, the T box system represents a target for antimicrobial agents. To date, it has not been possible to isolate this complex in a cell-free system in order to evaluate potential specific modulators or inhibitors of expression of T-box regulated genes. Thus, it would be useful to have a well-defined in vitro assay that would permit evaluation of agents that specifically interact with the T-box termination/antitermination complex. Such an assay system would permit the rapid and high throughput screening of potential inhibitors of expression of T-box regulated genes.